本文選題：鼻息肉病 + Th17細胞　； 參考：《重慶醫(yī)科大學》2013年博士論文

【摘要】：目的及背景：鼻息肉（nasal polyposis,NP）是一種鼻部黏膜的炎癥性疾病，在鼻科臨床上呈多發(fā)態(tài)勢，根據(jù)歐洲的鼻竇炎及鼻息肉治療指南（EP3OS）,鼻息肉被認為是慢性鼻-鼻竇炎（chronic sinusitis，CRS）的一個亞型。由于該病較高的復發(fā)率及目前治療手段無法達到完全根治該病，鼻息肉已成為嚴重降低人類生活質量并帶來大量社會經(jīng)濟負擔的全球性健康問題。 對NP發(fā)病機制的研究在近幾十年是鼻部炎癥性疾病研究領域的重點，國內外學者們對NP的起病原因及相關機制進行了大量的科學研究，盡管對于其疾病機理仍不能徹底地的予以明確，但是幾乎所有的研究均表明：鼻息肉的形成是一個多種因素參與、多步驟發(fā)展的過程，慢性持續(xù)性炎癥與變態(tài)反應與之關系密切。而Th淋巴細胞，尤其是Th17細胞在中國人鼻息肉的發(fā)病過程中扮演了重要的角色,而且變應性因素在中國人鼻息肉中很可能通過影響Th17細胞的反應發(fā)揮了關鍵作用。 作為一種關鍵的核轉錄因子，在胞外信號的作用下，STAT3的酪氨酸基團被JAK磷酸化，以二聚體的形式進入胞核內，與胞核中特異的基因片段相結合，調節(jié)下游基因及相關蛋白的表達。近年的研究發(fā)現(xiàn)STAT3信號通路在IL-6的刺激下參與Th17細胞的增殖活化，由STAT3介導下引起的Th17細胞反應在葡萄膜炎、克羅恩病、哮喘等免疫及炎癥性疾病的發(fā)病中發(fā)揮關鍵作用。那在中國，作為同樣是Th17細胞反應為主的NP中，STAT3信號通路的異常激活是否也存在呢？變應性因素對NP中Th17細胞反應的影響與其與NP中STAT3通路的活化狀態(tài)的關系有關聯(lián)嗎？ 為研究上述疑問，本研究以已經(jīng)明確診斷的NP患者作為實驗的對象，并將他們按照個體有無變應性的標準而分為atopic NP和non-atopicNP兩個組，以STAT3信號通路相關關鍵因子的異常激活為支撐點，研究分析在中國西南地區(qū)的漢族人群中，不同類型NP的局部炎癥特性與免疫機制，以解釋在atopic和non-atopic NP發(fā)病機制中STAT3信號通路所起的作用，同時探討變應性因素在NP中的作用與STAT3信號通路是否相關。本實驗將從新的研究途徑研究NP的發(fā)病機制，完善中國人NP是以Th17細胞反應為主這一理論，為NP的研究與治療提供新的方向與靶點。 第一部分STAT3信號通路相關因子在伴和不伴變應性體質鼻息肉患者中表達的研究 目的：通對檢測atopic和non-atopic NP兩組患者息肉組織中STAT3信號通路中代表激活的關鍵因子及其下游Th17細胞在RNA、蛋白水平的相關因子的表達。觀察兩組患者鼻腔鼻竇病變局部STAT3信號通路的異常激活狀態(tài)、Th17細胞反應狀態(tài)及兩組間的異同。旨在探討STAT3信號通路的異常激活在NP發(fā)病機制中的意義，及NP中變應性因素對Th17反應的影響與STAT3信號通路的異常激活之間的關系。 方法：對atopic-NP組20例，non-atopic NP組20例及對照的下鼻甲組織10例，采用免疫組化及Western Blot方法檢測兩組NP組織中pSTAT3及SOCS3的表達；采用ELISA檢測NP組織中IL-6、sIL-6R、IL-17的表達；采用real-time PCR檢測NP組織中RORc的表達；采用免疫熒光雙標方法檢測NP局部組織中CD4+RORc+細胞的表達。同時，以相關性分析的方法統(tǒng)計分析息肉組織中Th17細胞及其相關因子與STAT3信號通路的關鍵因子的關系。 結果：NP患者息肉組織中RORc、IL-17及CD4+RORc+細胞的水平比對照組明顯上升，在atopic NP組比另一NP組的表達更高，差距有統(tǒng)計學意義；與對照組相比，NP患者息肉組織中IL-6、sIL-6R、pSTAT3及SOCS3的表達顯著升高，且在atopic NP組pSTAT3的表達顯著高于non-atopic NP組，SOCS3的表達在atopic NP組顯著低于non-atopic NP組，而在這兩組NP中的IL-6與sIL-6R的水平的差異無統(tǒng)計學意義；在NP組織中，pSTAT3的表達水平與RORc、IL-17及CD4+RORc+細胞水平呈正相關，SOCS3的表達與pSTAT3的水平呈負相關，而IL-6與sIL-6R的水平與息肉組織中SOCS3的表達與pSTAT3的水平相關性無統(tǒng)計學意義。 結論：STAT3信號通路的異常激活及其下游Th17細胞反應的亢進存在于NP中，其在NP的發(fā)病機制中起到關鍵作用；且NP患息肉組織中STAT3信號通路關鍵因子與Th17細胞相關因子基本上消長一致，說明STAT3信號通路的異常激活在Th17細胞反應過度亢進過程中扮演關鍵角色；Atopic NP存在更為嚴重的STAT3信號通路的異常激活和更為嚴重的Th17細胞反應的亢進，說明變應性因素可能通過影響STAT3信號通路的異常激活推動Th17反應的進一步亢進，從而促進有更嚴重臨床病理特征的NP的發(fā)生發(fā)展。而SOCS3的表達差異可能是引起atopic NP組與non-atopic NP組出現(xiàn)IL-6及sIL-6R的水平與其下游的細胞因子的表達消長不統(tǒng)一的原因。 第二部分STAT3抑制劑阻斷鼻息肉中STAT3信號通路后相關下游因子表達的實驗研究 目的：通過不同濃度的STAT3信號通路抑制劑AG490作用于NP組織單細胞懸液，觀察阻斷NP中STAT3信號通路后對NP局部組織的Th17細胞及其相關因子的影響，并分析不同濃度下Th17細胞相關因子表達有無差異。以期在前一部分實驗基礎之上進一步探討STAT3信號途徑在NP發(fā)病中的作用，從體外研究的方面證明STAT3信號途徑在中國人NP的發(fā)病中扮演的關鍵角色。 方法：用不同濃度的STAT3通路抑制劑AG490體外作用培養(yǎng)于20例NP組織單細胞懸液（atopic NP10例，non-atopic NP10例）48h，根據(jù)濃度分為低濃度組（50μM）與高濃度組（100μM），以PBS代替AG490干預作為對照組。采取流式細胞術分析各組標本中Th17細胞的分布，運用real-time PCR檢測各組RORc的水平，以ELISA方法檢測各組中IL-17的水平。 結果：用AG490處理48h后，AG490作用組的Th17細胞及相關因子（RORc及IL-17）的表達較對照組顯著降低，差異有統(tǒng)計學意義；且的Th17細胞及相關因子（RORc及IL-17）的表達水平在高濃度AG490作用組較低濃度AG490作用組的水平有明顯降低，差異有統(tǒng)計學意義。 結論：在NP中STAT3信號通路的激活與Th17細胞的亢進反應密切相關，AG490阻斷STAT3活化可顯著下調RORc及IL-17的表達，，抑制NP中的Th17細胞反應。上述結果從體外實驗方面證實STAT3信號通路是通過影響Th17細胞反應來促進NP的發(fā)生發(fā)展，在NP的發(fā)病機制中發(fā)揮重要作用。 第三部分塵螨變應原體外刺激對鼻息肉患者STAT3信號通路相關因子局部表達的影響 目的：采用塵螨特異性變應原(HDM)作為刺激原，在體外短時期內刺激并培養(yǎng)患者NP組織單細胞懸液中的單個核細胞，觀察變應原體外刺激后，對兩組NP患者STAT3信號通路的影響，分析兩組間STAT3信號通路相關因子表達變化的差異，在前兩部分實驗基礎上進一步闡述變應性因素對兩組患者STAT3信號通路異常激活的影響，體外證實變應性因素是通過影響STAT3信號通路異常激活來促進TH17細胞的亢進反應從而在NP發(fā)病機制中發(fā)揮作用的。 方法：分別以大劑量HDM+PHA及單純PHA，對atopic和non-atopic NP患者局部NP組織單細胞懸液中細胞予以體外刺激培養(yǎng)48h，采用Western Blot方法檢測兩組NP組織中pSTAT3的表達，Real-time PCR方法及Western Blot方法檢測SOCS3的表達，并分析兩組間差異。 結果：HDM+PHA刺激后的NP組患者局部NP組織單細胞懸液細胞中pSTAT3及SOCS3的表達較對照組明顯上升，且在NP組內，變應性NP組的pSTAT3水平比非變應性NP組明顯上升，而變應性NP組SOCS3的表達水平則較非變應性NP組顯著下降；此外，在atopic NP組，HDM+PHA刺激使pSTAT3表達水平較PHA刺激顯著升高,而SOCS3的表達水平較PHA刺激顯著降低；而non-atopic NP組與對照組經(jīng)HDM+PHA刺激和經(jīng)PHA刺激對pSTAT3及SOCS3的表達水平影響差異無顯著性。 結論：伴變應性的NP患者的局部NP組織中HDM可誘導STAT3相關因子表達上調，揭示了STAT3信號通路在變應性上氣道炎癥中的重要作用，證實了變應性因素可通過影響STAT3信號通路在NP的發(fā)生發(fā)展中發(fā)揮作用；此外，在atopic NP組HDM誘導的STAT3相關抑制因子的表達下調，可以進一步支持前部分實驗中關于變應性因素影響STAT3相關抑制因子從而促進更嚴重的STAT3信號通路的異常激活的假設，在體外實驗方面解釋了atopic NP組與non-atopic NP組出現(xiàn)IL-6及sIL-6R的表達與其下游因子表達消長不一致的原因。
[Abstract]:Objective and background: nasal polyps (nasal polyposis (NP)) is an inflammatory disease of the nasal mucosa. It has a multiple trend in the clinic. According to the European nasosinusitis and nasal polyp treatment guidelines (EP3OS), nasal polyps are considered as a subtype of chronic nasosinusitis (chronic sinusitis, CRS). Due to the high recurrence rate and current treatment of the disease, the nasal polyps are considered as a subtype. Treatment can not completely cure the disease. Nasal polyps have become a global health problem that seriously reduces the quality of life of human beings and brings a lot of social and economic burden.
The research on the pathogenesis of NP has been the focus of the research field of the nasal inflammatory disease in recent decades. Scholars at home and abroad have carried out a lot of scientific research on the causes and related mechanisms of the onset of NP. Although the mechanism of its disease is still not completely clear, almost all studies have shown that the formation of nasal polyps is a one. Many factors participate in the process of multi step development, and chronic persistent inflammation and allergy are closely related. Th lymphocytes, especially Th17 cells, play an important role in the pathogenesis of Chinese nasal polyps, and the allergic factors are likely to play a key role in the reaction of Chinese nasal polyps by affecting the reaction of Th17 cells. Bond action.
As a key nuclear transcription factor, the tyrosine group of STAT3 is phosphorylated by JAK, entering the nucleus in the form of two polymer, combining with the specific gene fragments in the nucleus and regulating the expression of the downstream genes and related proteins. In recent years, the present STAT3 signaling pathway is involved in Th17 fines under the stimulation of IL-6. Cell proliferation activation, the Th17 cell response induced by STAT3 plays a key role in the pathogenesis of uveitis, Crohn's disease, asthma and other immune and inflammatory diseases. In China, is the abnormal activation of STAT3 signaling as the same Th17 cell response NP? Allergic factors to Th17 cells in NP Is there any relationship between the effect of reaction and the activation state of STAT3 pathway in NP?
In order to study the above questions, the NP patients who have been clearly diagnosed are selected as the subjects of the experiment, and they are divided into two groups of atopic NP and non-atopicNP according to the standard of the individual unaltered, and the abnormal activation of the key factors of the STAT3 signaling pathway is the support point. The local inflammatory properties and immune mechanisms of the same type of NP are used to explain the role of STAT3 signaling pathway in the pathogenesis of atopic and non-atopic NP, and to explore whether the role of the allergic factors in NP is related to the STAT3 signaling pathway. This experiment will study the pathogenesis of NP from a new approach and improve the NP in China by Th17 cells. This theory provides a new direction and target for the research and treatment of NP.
Part one: the expression of STAT3 signaling pathway related factors in patients with and without allergic rhinitis.
Objective: To investigate the key factors that represent the activation of the STAT3 signaling pathway in the polyps of atopic and non-atopic NP two groups and the expression of related factors in the level of RNA and protein in the downstream Th17 cells. The abnormal activation state of the local STAT3 signaling pathway in the two groups of nasal sinuses, the reaction state of Th17 cells and the two groups were observed. The purpose of this study is to explore the significance of abnormal activation of STAT3 signaling pathway in the pathogenesis of NP, and the relationship between the effect of allergic factors on the response of Th17 and the abnormal activation of the STAT3 signaling pathway in NP.
Methods: 20 cases in group atopic-NP, 20 cases in group non-atopic NP and 10 cases of inferior turbinate tissue in control, the expression of pSTAT3 and SOCS3 in two groups of NP tissues were detected by immunohistochemistry and Western Blot, and the expression of IL-6, sIL-6R, and SOCS3 in the tissues of NP was detected by ELISA. A double standard method was used to detect the expression of CD4+RORc+ cells in the local tissue of NP, and the correlation analysis was used to analyze the relationship between the Th17 cells and their related factors in polyp tissues with the key factors of the STAT3 signaling pathway.
Results: the levels of RORc, IL-17 and CD4+RORc+ cells in the polyp tissues of NP patients were significantly higher than those in the control group, and the expression in atopic NP group was higher than that in the other NP group, and the difference was statistically significant. In the group of non-atopic NP, the expression of SOCS3 in the atopic NP group was significantly lower than that in the non-atopic NP group, but there was no significant difference in the level of IL-6 and sIL-6R in the two groups of NP. There was no significant correlation between the level of R and the expression of SOCS3 in polyp tissue and pSTAT3 level.
Conclusion: the abnormal activation of the STAT3 signaling pathway and the hyperactivity of the downstream Th17 cell reaction exist in NP, which plays a key role in the pathogenesis of NP, and the key factor of STAT3 signaling pathway in NP polyps with Th17 cell related factors is basically the same, indicating that the abnormal activation of the STAT3 signaling pathway is overreactive in Th17 cells. Hyperactivity plays a key role in the process of hyperactivity; Atopic NP has a more serious STAT3 signaling pathway and a more severe Th17 cell response, suggesting that the allergic factors may promote the further hyperactivity of the Th17 reaction by affecting the abnormal activation of the STAT3 signaling pathway, thus promoting a more severe clinical pathological NP. The difference in expression of SOCS3 may be the reason why the level of IL-6 and sIL-6R in the group atopic NP group and the non-atopic NP group and the expression of the downstream cell factors are not uniform.
The second part is the experimental study of STAT3 inhibitor blocking the expression of downstream related factors after STAT3 signaling pathway in nasal polyps.
Aim: To observe the effect of blocking the STAT3 signaling pathway in NP on the Th17 cells and its related factors in the local tissues of NP, and to analyze the difference in the expression of Th17 cells related factors at different concentrations after the inhibition of the STAT3 signaling pathway inhibitor AG490 on the single cell suspension of NP tissue, and to analyze the difference in the expression of the related factors of Th17 cells under different concentrations. Objective to explore the role of STAT3 signaling pathway in the pathogenesis of NP, and to demonstrate the pivotal role of STAT3 signaling pathway in the pathogenesis of NP in vitro.
Methods: 20 cases of NP tissue single cell suspension (atopic NP10, non-atopic NP10 case) 48h were cultured with different concentration of STAT3 pathway inhibitor AG490 in vitro. The concentration was divided into low concentration group (50 micron) and high concentration group (100 mu M), and PBS instead of AG490 intervention was used as the control group. The levels of RORc in each group were detected by real-time PCR, and the level of IL-17 in each group was detected by ELISA.
Results: after the treatment of 48h with AG490, the expression of Th17 cells and related factors (RORc and IL-17) in the AG490 group was significantly lower than that of the control group, and the difference was statistically significant. The levels of Th17 cells and related factors (RORc and IL-17) were significantly lower in the lower concentration AG490 action group of the high concentration AG490 action group, and the difference was statistically significant. Learning meaning.
Conclusion: the activation of STAT3 signaling pathway in NP is closely related to the hyperactivity of Th17 cells. AG490 blocking the activation of STAT3 can significantly reduce the expression of RORc and IL-17 and inhibit the Th17 cell response in NP. These results confirm that the STAT3 signaling pathway promotes the occurrence and development of NP by influencing Th17 cell response in vitro. It plays an important role in the mechanism of disease.
The third part is the influence of dust mites in vitro stimulation on local expression of STAT3 signaling pathway related factors in patients with nasal polyps.
Objective: to use dust mite specific allergens (HDM) as a stimulator, to stimulate and cultivate mononuclear cells in a single cell suspension of NP tissue in short period in vitro, to observe the effect of allergen to the STAT3 signaling pathway in two groups of NP patients in vitro, and to analyze the difference in the expression of STAT3 signaling pathway related factors between the two groups, in the first two On the basis of some experiments, the effect of allergic factors on the abnormal activation of STAT3 signaling pathway in two groups of patients was further elaborated. In vitro, the allergic factors were proved to play a role in the pathogenesis of NP by affecting the abnormal activation of the STAT3 signaling pathway to promote the hyperactivity of the TH17 cells.
Methods: the cells in the local NP tissue suspension of atopic and non-atopic NP were stimulated and cultured in vitro by large dose of HDM+PHA and simple PHA. The expression of pSTAT3 in the two groups of NP tissues was detected by Western Blot method. The expression of the two groups was detected and the differences between the groups were analyzed.
Results: the expression of pSTAT3 and SOCS3 in the single cell suspension cells of group NP after HDM+PHA stimulation was significantly higher than that in the control group, and in the NP group, the pSTAT3 level in the allergic NP group was significantly higher than that in the non allergic NP group, while the SOCS3 expression level in the allergic NP group was significantly lower than that in the non allergic NP group. The expression level of pSTAT3 was significantly higher than that of PHA stimulated by HDM+PHA stimulation, while the expression level of SOCS3 was significantly lower than that of PHA, but there was no significant difference between the non-atopic NP group and the control group by HDM+PHA stimulation and PHA stimulation on the expression level of pSTAT3 and SOCS3.
Conclusion: HDM can induce the up-regulated expression of STAT3 related factors in the local NP tissues of patients with allergic NP, which reveals the important role of STAT3 signaling pathway in allergic airway inflammation, and confirms that the allergic factors can play a role in the occurrence and development of NP by affecting the STAT3 signaling pathway; furthermore, STAT3 in atopic NP group HDM induces STAT3. Down regulation of related inhibitory factors can further support the hypothesis that the STAT3 related factors affect the abnormal activation of the more serious STAT3 signaling pathway in the previous experiments. In vitro experiments, the expression of IL-6 and sIL-6R in the atopic NP group and the non-atopic NP group and the downstream factor table are explained. The cause of the disagreement.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R765.25

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